Mammalian blood cells are an attractive target for manipulation by genetic engineering. Many blood diseases are caused by defects in single genes, and these diseases could be treated through gene therapy by insertion of a single correct gene copy in appropriate cells. Examples of single gene defects are hemophilias, such as Factor IX deficiency and Factor VIII deficiency, and immuno-deficiencies, such as adenosine deaminase (ADA) deficiency. Manipulation of blood cells by the addition of a normal or corrective gene copy would provide a therapeutic strategy for treatment of these diseases. Blood cells are one of the ideal candidates for delivery of peptides or proteins systemically since they can secrete these products into the blood circulation. Genetic manipulation of blood cells in non-human animals may also be useful by providing experimental animal models for development of clinical protocols.
Lymphocytes have previously been the subject of genetic manipulation. Lymphocytes arise from the lymphoid system and comprise 20% of all leucocytes (white blood cells). During exposure to an antigen, specific lymphocytes are stimulated by the antigen. Stimulated lymphocytes may proliferate and produce antibodies to the antigen or may become part of a cellular immune response. The two major types of lymphocytes are T cells, which become helper or killer cells and are responsible for cellular immune response, and B cells, which produce antibodies.
In one prior example of genetic manipulation of lymphocytes, tumor infiltrating lymphocytes (TIL) have been isolated from melanoma tumors, infected with a retrovirus vector, and returned to patients. Rosenberg, et al., N. Eng. J. Med. 323: 570-578 (1990). The TILS were infected with the retrovirus simply to mark them so that their fate in the patient could be monitored. The study determined that the infused TILS persisted in the patient and produced no adverse effect. Recently, genetically transformed T and B cells have been proposed as a treatment for ADA deficiency. The T and B cells from ADA deficient patients would be infected with a retrovirus vector encoding an ADA gene, and these infected cells returned to the patient. Canto, et al., Proc. Natl. Acad. Sci. USA 83:6563-6567 (1986).
Bone marrow cells are another attractive target for genetic manipulation. Hematopoietic stem cells found in the bone marrow produce all the cells present in blood--lymphocytes, erythrocytes, platelets, granulocytes, macrophages and monocytes. Mitotic division of the stem cells produces two daughter cells, which either return to the stem cell pool or differentiate into a specific type of blood cell. Differentiation of stem cells involves consecutive cell differentiation and ends with the creation of various defined blood cell populations which live for up to a few months and then die.
The hematopoietic system is an attractive target for gene transfer for several reasons. First, well-developed procedures exist for bone marrow transplantation. Second, hematopoietic cells develop into many different kinds of cells, and there are many genetic diseases that affect these blood cells.
Gene transfer into cells at different stages in the hematopoietic system will have different results. Transformed differentiated cells will express the gene transiently in a certain type of cell for a limited time--until the cell dies. Transformation of a stem cell can result in a continued stable expression of the gene, in all of the cells derived from that stem cell, for the life of the animal.
Several research groups have demonstrated gene transfer into hematopoietic stem cells of mice by procedures different than those of the present invention. A. D. Miller, Blood 76[2]: 271-278 (1990), describes a typical stem cell experiment. Donor animals were first treated with 5-fluorouracil to kill differentiated blood cells. This treatment was intended to induce the mitotic division of stem cells. Retrovirus vectors, which are effective for transformation only in dividing cells, were then exposed to the cells. The putatively transformed bone marrow was then injected into recipient animals. Recently, several groups have shown long-term expression of both the human beta-globin and the ADA gene in mice using the retrovirus procedure.
Miller (above, at 273) details some of the current problems in bone marrow genetic transformation. One particular problem is that "much of the repopulating ability of marrow is lost during the infection procedure." Miller points out that in applications where donors are limited, such as in humans, such losses may be a considerable practical obstacle to gene therapy.
What is needed in the art of gene transfer is an effective method of transforming unattached cells such as blood and hematopoietic cells. Previously, the vast majority of efforts directed at transformation of unattached cells have used retrovirus transformation vectors or electroporation. The apparatus used for the transformation technique of the present invention is based on a quite different method of transporting the foreign DNA into the genome of the target cells. As disclosed by Klein et al., Nature, 327: 70-73 (1987), an instrument for the acceleration of very small particles of metal, coated with DNA, is effective in causing transient expression in plant cells in vivo. The transforming DNA is coated onto very small particles which are shot as ballistic projectiles into the tissues to be transformed. While the apparatus described by Klein, et al. has been demonstrated to have utility in transforming plant cells in culture, this particular apparatus has the disadvantage that the force of particle impact is not readily adjustable. Thus, it is a difficult apparatus to use for transformation of different cells and organisms, because a wide range of kinetic energies of particle propulsion are not available. Yang, et al. (Proc. Natl. Acad. Sci. 87: 9568-9572 (December, 1990)) disclose a method of transforming solid tissue mammalian somatic cells in situ via particle bombardment. Yang, et al. employed a particle acceleration device with an adjustable voltage and transformed cell cultures and liver, skin and muscle tissues. A similar device is illustrated as effective in germ line transformation of plants in U.S. Pat. No. 5,015,580.